Process for producing (r)-3-hydroxybutyl (r)-3-hydroxybutyrate

ABSTRACT

Embodiments of the present invention are directed to processes for the production of (R)-3-hydroxybutyl (R)-3-hydroxybyrate. Poly (R)-3-hydroxybyrate is transesterified with an alcohol, to form a first ester portion and a second ester portion. The first ester portion is reduced to the diol to form a diol portion and the diol portion is reacted with the second ester portion to produce (R)-3-hydroxybutyl (R)-3-hydroxybyrate.

The invention relates to a process for producing (R)-3-hydroxybutyl(R)-3-hydroxybutyrate. In particular, the invention relates to a processfor producing (R)-3-hydroxybutyl (R)-3-hydroxybutyrate from a singlestarting material feedstock of poly-(R)-3-hydroxybutyrate.

Ketone bodies are chemical compounds which are produced by the liverfrom fatty acids released from adipose tissue. Ketone bodies themselvescan be used as a source of energy in most tissues of the body. Theintake of compounds that boost the levels of ketone bodies in the bloodcan lead to various clinical benefits, including an enhancement ofphysical and cognitive performance and the treatment of cardiovascularconditions, diabetes, neurodegenerative diseases and epilepsy. Ketonebodies include (R)-3-hydroxybutyrate and acetoacetate.

WO2004/108740 discloses that ketone bodies may be administered directlyto achieve elevated levels of ketone bodies in a subject. However,direct administration of the compounds is unpractical and potentiallydangerous. For example, direct administration of either(R)-3-hydroxybutyrate or acetoacetate in its free acid form can resultin significant acidosis following rapid absorption from thegastrointestinal tract. Administration of the sodium salt of thesecompounds in unregulated amounts is also unsuitable due to a potentiallydangerous sodium overload that could accompany administration oftherapeutically relevant amounts of the compounds. Examples of thederivatives include esters, for instance esters derived from a varietyof alcohols and oligomers of (R)-3-hydroxybutyrate.

WO2010021766 discloses that one particular enantiomer of one particularester of 3-hydroxybutyrate is an effective and palatable precursor tothe ketone body (R)-3-hydroxybutyrate. Thus WO2010021766 discloses3-hydroxybutyl 3-hydroxybutyrate enantiomerically enriched with respectto (R)-3-hydroxybutyl (R)-3-hydroxybutyrate.

Various synthetic approaches have been developed for the production ofthis stereoisomer. Methods are known for producing hydroxybutyrate frompoly-(R)-3-hydroxybutyrate but involve a large number of steps and arecomplex. Other synthetic approaches have been attempted but have varioustechnical and commercial drawbacks including low yields, the productionof impure product, impracticability on a large scale and cost.

WO2010/120300 discloses various methods of a producing(R)-3-hydroxybutyl (R)-3-hydroxybutyrate involving enantioselectivereduction of a compound of formula I, II or III.

WO2010/120300 also discloses a process involving treating HOCH₂CH₂COCH₃with a diketene of formula VI in WO2010/120300, CH2═C(CH2)—O—C═O andsubjecting the reaction to enantioselective reduction. Further processesinvolving treating butane-1,3-diol with the ketene VI withenantioselective reduction and a process starting from 4-hydroxybutanoneare also disclosed. The enantioselective reduction is carried out usinga ketoreductase or alcohol dehydrogenase.

Whilst effective at producing (R)-3-hydroxybutyl (R)-3-hydroxybutyratethese starting materials may be costly and higher rates of reaction maybe desirable. There remains a need to be able to produce(R)-3-hydroxybutyl (R)-3-hydroxybutyrate at higher volumes and toimprove the economics of production.

We have now found that these problems may be addressed by subjectingpoly-(R)-3-hydroxybutyrate, a relatively low cost starting material, toa process that involves transesterification divides the startingmaterial or feedstock into two portions or streams, producing a reducedintermediate from a first portion or stream, which is then reacted witha second portion or stream to provide (R)-3-hydroxybutyl(R)-3-hydroxybutyrate. in a first aspect the invention provides aprocess for the production of (R)-3-hydroxybutyl (R)-3-hydroxybutyratecomprising:

-   -   (i) contacting poly-(R)-3-hydroxybutyrate with an alcohol to        transesterify the poly-(R)-3-hydroxybutyrate under        transesterification conditions to produce an ester of        (R)-3-hydroxybutyrate and the alcohol;    -   (ii) separating the product of step i) into a first and second        portion and reducing the first portion of the        (R)-3-hydroxybutyrate ester to form (R)-1,3-butanediol;    -   (iii) contacting under transesterification conditions the        (R)-1,3-butanediol from step ii) with the second portion of the        transesterified ester to produce (R)-3-hydroxybutyl        (R)-3-hydroxybutyrate.

The process allows industrial scale production of enantiomericallyenriched monoester of (R)-3-hydroxybutyric acid and (R)-1,3-butanediolfrom bulk poly-(R)-3-hydroxybutyrate which is commercially available inlarge scale and acceptable cost, for example by fermentation of cornstarch or sugar cane.

The term “enriched”, as employed herein, means that the level of theenriching isomer is higher than the level at which that isomer would bepresent in a racemic mixture. Where a percentage enrichment is referredto, the enriching isomer constitutes that molar percentage of the total3-hydroxybutyl 3-hydroxybutyrate product present.

Preferably enantiomeric purity is measured using chiral high performanceliquid c h omatography (chiral HPLC). Measurements are typically madeagainst the corresponding racemic mixture. Alternatively, chiral gas c homatography (chiral GC) may be used reliably. Accordingly, where apercentage enrichment is referred to herein, the percentage enrichmentis typically that measured by chiral HPLC or by chiral GC. Preferably,the percentage enrichment is that measured by chiral HPLC. Usually, theenzyme employed is one which is capable of reducing said compound offormula (II), (III) or (IV) to produce 3-hydroxybutyl 3-hydroxybutyratewhich is enantiomerically enriched to at least 95%, for instance to atleast 97%, to at least 98%, or to at least 99%, with respect to(R)-3-hydroxybutyl(R)-3hydroxybutyrate.

The process may be continuous or batch. Advantageously, the inventionenables a high t h oughput industrial production of (R)-3-hydroxybutyl(R)-3-hydroxybutyrate from poly-(R)-3-hydroxybutyrate which may beobtained from corn starch.

Preferably, the poly-(R)-3-hydroxybutyrate feedstock is provided from asingle feedstock by fermentation of corn starch with microorganisms.

The poly-(R)-3-hydroxybutyrate feedstock may be transesterified in stepi) using any suitable alcohol which allows the formed ester to bereduced to (R)-1,3-butanediol. Suitably a dihydric or trihydric alcoholis employed but preferably the alcohol is monohydric, for example a C1-6alcohol. Where (R)-3-hydroxybutyl (R)-3-hydroxybutyrate is forconsumption for example as a food or nutritional supplement, the alcoholis suitably ethanol as this is more acceptable for consumption thanother alcohols.

Suitably, the alcohol is present in sufficient quantity thatpoly-(R)-3-hydroxybutyrate moieties may be esterified. Preferably theweight ratio of alcohol to poly-(R)-3-hydroxybutyrate is from 1:1 to10:1, more preferably from 2:1 to 6:1.

The transesterification in step i) is suitably carried out in acidicconditions. Preferably, the reaction mixture comprises an acid catalyst.The acid may be organic or inorganic and is preferably a mineral acid,for example sulphuric acid. The catalyst may be solid as desired.

Suitably the transesterification is carried out at elevated temperature,preferably greater than 50° C., greater than 90° C. and desirably notmore than 150° C. Elevated pressure may be employed. Suitably, thetransesterification is carried out for sufficient time to affecttransesterification to an economically acceptable degree having regardto the temperature, catalyst and alcohol employed. Preferably, thetransesterification step is carried out for at least 1 hour, morepreferably at least 10 hours, and especially 15 to 30 hours, for example20 hours, 22 hours and 24 hours.

The product of the transesterification reaction may then be treated byone or more optional steps including filtering, purification, forexample by distillation and neutralisation for example by the additionof base for example hydroxide, bicarbonate and acetate, particularlycalcium hydroxide or sodium bicarbonate to neutralise the acid present.

Suitably, the ester of (R)-3-hydroxybutyrate is separated from thereaction mixture by removal of alcohol and optionally by-products of thereaction. The separation may be carried out in multiple stages asdesired. In a preferred embodiment, the ester is separated and purifiedfrom the alcohol and reaction by-products, The ester may be separatedfrom unreacted alcohol and other undesired materials, for example alkylcrotonate by separation of the liquid phase, for example by distillationof the alcohol and alkyl crotonate. The alcohol and by-products may beremoved by multiple distillations, suitably at atmospheric pressure andat a temperature above the boiling point of the alcohol, for examplegreater than 80° C., greater than 110° C. for example at a temperatureof 110 to 150° C. The ester of (R)-3-hydroxybutyrate is then suitablyseparated to provide a first portion which is subjected to a reductionreaction. The reduction in step ii) may be a hydride transfer reduction,hydrogenation, hydrosilylation followed by silyl ether hydrolysis.Preferably the reduction is carried out with any suitable reducing agentfor reducing an ketoester. The reducing agent may be organic orinorganic. The reduction step may be mediated by an enzyme, for examplea ketoreductase (KRED) or an alcohol dehydrogenase (ADH), and may benaturally occurring or commercially available, for example as describedin WO2010/120300.

The reducing agent may comprise hydrogen and a hydrogenation catalystmay be employed, for example Raney nickel, desirably employed atelevated pressure and temperature and catalysts comprising platinum,palladium, rhodium, iridium or ruthenium. Preferably the reducing agentemploys a hydride transfer reagent. Examples of suitable reducing agentsinclude complex metal hydrides for example, lithium aluminium hydride,lithium tetrahydridoaluminate, sodium bis (2-methoxyethoxy) aluminiumhydride, sodium borohydride, nickel borohydride, other inorganicreducing agents, for example, sodium hydrosulphite, sodiumtetrahydroborate and ruthenium hydrogenation catalysts known in the art,for example ruthenium hydride and rhodium hydrogenation catalysts knownin the art, aluminium triisopropoxide, and organic reducing agentsincluding a chiral borane, for example 2,5-dimethylborolane,borontrihydride:tetrahydrofuran or caticholborane, and enzymes andcofactors, for example nicotinamide, adenine dinucleotide (NADH) andnicotinamide adenine dinucleotide phosphate (NADPH). As desired acofactor recycling system is suitably employed.

The reducing step is suitably carried out under reducing conditions. Asolvent may be employed. The solvent may be anhydrous, for examplediethyl ether or tetrahydrofuran, or may be carried out in polar proticsolvent, for example water, alcohol and basic aqueous media, dependingupon the reducing agent.

Preferably, the reducing step is carried out in aqueous solution and amoderately strong reducing agent is employed so as to ensure retentionof the desired stereochemistry. Desirably, the temperature of thereducing step is controlled to avoid significant temperature rise, andis desirably carried out at a temperature below standard temperature,desirably under 10° C., for example −5 to 3 C.

Suitably, the reducing agent is contacted with the first portion slowlyto avoid undue temperature rise. The reducing agent and first portionare suitably allowed to react over an extended period of time, forexample at least 30 minute, preferably at least 1 hour, more preferably1 to 20 hours, especially 4 to 10 hours. Upon completion of thereduction reaction to the desired degree, the reaction may be quenchedby addition of a quenching agent, for example by addition of acid, forexample sulphuric acid and allowed to stand for a period of time, forexample at least 1 hour, preferably 1 to 20 hours, for exampleovernight. Thereafter, the reaction mixture may be contacted with aremoval agent, for example hydroxide and especially calcium hydroxide toremove salts of the reducing agent and quenching agent.

The butanediol produced from the first portion is then contacted withthe second portion of the ester of (R)3-hydroxybutanoate.

The transesterificatioin is suitably carried out in the presence of atransesterification catalyst, for example an enzyme, acid or base.Suitable examples of enzymes include lipase, examples of suitable acidsinclude mineral acids for example sulphuric acid and hydrochloric acid,examples of suitable bases include alkali metal hydroxides and alkalimetal alkoxides.

Preferably, the transesterification reaction between the second portionand (R)-1,3-butanediol is carried out at elevated temperature, forexample from 30 to 150° C., particularly 40 to 100° C.

This transesterification process may be carried out in a batch orcontinuous process.

Suitably the transesterification process is carried out for at least 1hour, preferably 1 to 20 hours, for example 5 to 10 hours. Uponcompletion of the reaction to the desired degree, the product of thereaction may then be subjected to further treatment to remove catalyst,unreacted starting materials and by-products, for example by filtering,distillation or the like.

The invention will be illustrated by the following non-limitingexamples.

Example 1

Transesterification Step i)

A 5 gallon Parr reactor is charged with 12.5 L(10 kg) absolute ethanoland 2.5 kg poly (R)-3-hydroxybutanoate (Biocycle, Fazenda de Pedra, cPostal 02 CEP 14158-00, Serenaa, S. P. Brazil) and stirred for 2-5min tocomplete mixing after which 0.1 L concentrated sulfuric acid is addedslowly to the mixture, The mixture is heated with a 300° C./h ramp to110° C. and the reactor held in soak mode for a total run time is 22 h.The unit is cooled to about 30° C. using chilled water. After thetemperature has fallen below 60° C., the digester is vented and purgedwith nitrogen to remove formed ether. An amount of base, equal to theequivalents of acid is added to the crude digest with stirring toneutralize the acid. Stirring is continued about 16 h after which thestirring is stopped and the solids left to settle. The liquid phase issiphoned off into a wiped film distillation apparatus and distilled inphases to remove first the ethanol and ethyl crotonate (a side product),and then the ethyl (R)-3-hydroxybutyrate. Ethanol/ethyl crotonate isdistilled off over 3 passes generally at atmospheric pressure and bandheater and pump flow rates of 120 & 5 L/h, 120 & 3 L/h and 140 & 3 L/hrespectively. The ethyl (R) 3-hydroxybutyrate is distilled at 10 mmHg,band heater=88 and feed rate 4 L/h. The primary chiller is set to 5° C.and the secondary chiller at −1° C. for all distillations. The trap ischarged with dry ice and either acetone or IPA. When collecting theethyl (R) 3-hydroxybutyrate, the residue from the first pass is recycledthrough the still to recover more product. Theethyl-(R)-3-hydroxybutyrate is assayed by GC-MS and NMR for purity.

Reduction Step ii)

A heavy duty stainless steel stock pot is charged with 12 L water and aportion of (3.49 L) ethyl (R) 3-hydroxybutyrate. Both water and esterwere previously chilled to 4° C. for at least 24 h. The stock pot issurrounded by ice, gassed with nitrogen and stirred. After about 1 h, 1Kg sodium borohydride is added in small aliquots to order to minimizetemperature gain. Borohydride addition takes about 1 h and thetemperature should be kept below 20° C. during the NaBH₄ addition. About5 h after borohydride addition the reaction is quenched by slowly adding745 ml concentrated sulfuric acid. The mixture is allowed to stand, withstirring overnight and the temperature rise to room temperature. Themixture is filtered, the filtrate heated to 90° C. and neutralized* byadding calcium hydroxide with stirring. After 2 hours mixture is cooledand filtered and the filtrate ionic strength reduced using ion exchangeresins after which the solution is placed on a Buchi Rotovap and thebulk of the water removed. This leaves a viscous liquid assaying to >10M (R) 1,3-butanediol and containing 5-10% water. Remaining water isremoved by nitrogen purge or distillation. The purity is checked byenzymatic assay, GC-MS and NMR.

Transesterification Using R-1,3-butanediol

A solution is prepared by combining and mixing 600 ml of (R)1,3-butanediol and 1200 ml of ethyl (R)-3-hydroxybutanoate in astainless steel pan. A nylon mesh “tea bag” containing lipase is laid inthe solution and the pan is placed on a heating pad set to 40° C. The“tea bag” is sewn with lanes to keep the enzyme dispersed. The reactionis carried out under nitrogen with agitation. After 6 h the reaction isstopped by removing the “tea bag” and collecting the solution. Thesolution is passed through a filter to remove any enzyme resin “fines”and collected. Once enough crude solution has been collected thesolution is distilled sequentially to first degas and remove anyremaining ethanol, then to remove ethyl (R)-3-hydroxybutanoate, (R)1,3-butanediol and finally to collect the desired pure ketone ester,(R)-3-hydroxybutyl (R)-3-hydroxybutyrate. Recovered ethyl(R)-3-hydroxybutanoate and (I) 1,3-butanediol are recycled in subsequenttransesterification experiments. Crude solutions and still fractions areanalyzed by GC-MS.

1. A process for the production of (R)-3-hydroxybutyl(R)-3-hydroxybutyrate comprising: (i) contactingpoly-(R)-3-hydroxybutyrate with an alcohol to transesterify thepoly-(R)-3-hydroxybutyrate under transesterification conditions toproduce an ester of (R)-3-hydroxybutyrate and the alcohol; (ii)separating the product of step i) into a first and second portion andreducing the first portion of the (R) 3-hydroxybutyrate ester to form(R)-1,3-butanediol; (iii) contacting under transesterificationconditions the (R)-1,3-butanediol from step ii) with the second portionof the transesterified ester to produce(R)-3-hydroxybutyl-(R)-hydroxybutanoate.
 2. A process according to claim1 wherein the poly-(R)-3-hydroxybutyrate is obtained from corn starch orsugar cane.
 3. A process according to claim 1 wherein thepoly-(R)-3-hydroxybutyrate is transesterified in step i) using ethanol.4. A process according to claim 1 wherein the weight ratio of alcohol topoly-(R)-3-hydroxybutyrate is from 1:1 to 10:1.
 5. A process accordingto claim 1 wherein the step i) is carried out in acidic conditions.
 6. Aprocess according to claim 1 wherein the product of step i) is treatedto neutralise the acid, remove the alcohol by distillation.
 7. A processaccording to claim 6 wherein the distillation is carried out at atemperature of 110° C. to 150° C.
 8. A process according to claim 1wherein the reduction in step ii) comprises reducing(R)-3-hydroxybutyrate ester using a hydride transfer reagent.
 9. Aprocess according to claim 8 wherein the reducing agent is selected fromlithium aluminium hydride, sodium bis (2-methoxyethoxy) aluminiumhydride, sodium borohydride, nickel borohydride.